Polymorphism of human platelet membrane glycoprotein iiia and diagnostic and therapeutic applications thereof

ABSTRACT

Isolated polynucleotide molecules, and peptides encoded by those molecules, can be used in the analysis of alloantigen phenotypes, as well as in diagnostic and therapeutic applications relating to human platelet Pl A  polymorphism. In this vein, a method for typing blood cell and platelet membrane glycoproteins entails an analysis of amplified cDNA, encoded by platelet and red blood cell mRNA.

BACKGROUND OF THE INVENTION

The present invention relates to isolated polynucleotide moleculesuseful for analyzing alloantigen phenotypes, to peptides encoded bythese molecules, and to the diagnostic and therapeutic uses thereofrelating to human platelet Pl^(A) polymorphism, including a method fortyping blood cell and platelet membrane glycoproteins which entails ananalysis of amplified cDNA encoded by platelet and red blood cell mRNA.

Blood obtained from different individuals has been found to havedifferent antigenic and immune properties, to the extent that antibodiesin the blood of one person may react with antigens on red blood cells orplatelets in the blood of another individual. These antigens are oftenfound on membrane glycoproteins present on the surface of the cellmembranes. These membrane glycoprotein antigens can induce theproduction of antibodies against them when they are introduced asforeign proteins in transfused blood or in fetal blood. Human plateletsand red blood cells contain dozens of identifiable membrane glycoproteinconstituents, only some of which have been well characterized.

Membrane glycoproteins which induce antibody production in the samespecies are called "alloantigens." Alloantigens have been characterizedfor both red blood cells and platelets. Recognized classes of red bloodcell and platelet alloantigens have been described, over the past 30years, based on observations of antibody reactions occurring whenpatients have been exposed to blood from other individuals. The lack ofsequenceable antigen protein and clonable antigen-encoding mRNA hasprevented molecular characterization of the different alleles coding formany clinically important alloantigens.

One system of alloantigens, consisting of the platelet PlAI and PlA2alloantigens, are carried by the human platelet membrane glycoproteinIIb-IIIa (GPIIb-GPIIIa) complex, which mediates platelet aggregation byproviding functional receptors for fibrinogen on platelet surfaces. SeePhillips et al., Blood 71: 831-43 (1988). GpIIb and GpIIIa are known tobear a number of clinically important, alloantigenic determinants whichare responsible for eliciting an immune response in two well-describedclinical syndromes, post-transfusion purpura (PTP) and neonatalalloimmune thrombocytopenia (NATP). See Kunicki & Newman in CURRENTSTUDIES IN HEMATOLOGY AND BLOOD TRANSFUSION 18-32 (1986); Aster inADVANCES IN IMMUNOLOGY AND BONE MARROW TRANSPLANTATION 103-118 (1984).

The alloantigen system most frequently implicated in these disorders isthe Pl^(A) alloantigen system. There are two serologically defined, butmolecularly undefined, allelic forms of the Pl^(A) alloantigen,designated "Pl^(A1) " and "Pl^(A2)," which are thought to be expressionproducts of the GPIIIa gene. Kunicki & Newman, id., at 18-32. The genefrequencies for these two alleles have been calculated to be 85% for A1and 15% for A2, see Shulman et al., J. Clin. Invest 40: 1597-620 (1961).Since 98% of the population carries the Pl^(A1) antigen, individuals whoare Pl^(A2) homozygotes are at risk of producing anti-Pl^(A1) antibodiesagainst paternally-inherited Pl^(A1) antigens present on fetalplatelets, and are most likely to develop PTP following bloodtransfusion.

Determination of the amino acid sequence variations that are presumablyresponsible for forming the relevant epitopes of red blood cell andplatelet alloantigens has been achieved in only a few instances, duelargely to the formidable difficulties in obtaining protein-sequenceinformation from those often large glycoproteins. For example, theamino-acid sequence variation responsible for the relevant epitopes hasnot yet been reported for either the Pl^(A1) or Pl^(A2) forms of the 100kilodalton (kd) GPIIIa molecule. In this regard, there is considerabledata to support the notion that platelets and red blood cells, sincethey are anucleate cells, possess only vestigial amounts ofprotein-synthetic capability, although specific protein biosynthesis hasbeen demonstrated in platelets, see Plow, Thromb. Haemostasis 42: 666-78(1979); Kieffer et al, Eur. J. Biochem. 164: 189-95 (1987); Belloc etal, Biochim. Biophys. Acta. 925: 218-25 (1987), and in newly formed redcells (reticulocytes). Thus, it has not been considered feasible toobtain platelet or red blood cell cDNA libraries, thereby to analyzealloantigen-encoding sequences and determine the molecular basis of thealloantigen phenotypes.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provide DNAmolecules that can be used in analyzing an alloantigen phenotype fromplatelets and red blood cells.

It is also an object of the present invention to provide methodology fortyping membrane glycoproteins of human platelet and red blood cells,based on information obtained through the analysis of cDNA discovered tobe producible from anucleate-cell (platelet and red blood cell) mRNA.

It is yet another object of the present invention to provide ready meansfor determining platelet Pl alloantigen phenotypes.

It is still a further object of the present invention to providepolypeptide molecules useful in generating antibodies that distinguishbetween the alleles of different platelet membrane qlycoproteins

Another object of the present invention is to provide methods fordiagnosing and treating certain clinical syndromes related to an immuneresponse associated with platelet membrane glycoprotein alloantigens.

In achieving these objects, there has been provided, in accordance withone aspect of the present invention, an isolated DNA molecule thatcomprises a DNA sequence comprising at least nucleotide 196 of GPIIIa.In a preferred embodiment, nucleotide 196 comprises a cytosine or athymine base.

In accordance with another aspect of the present invention, a method hasbeen provided for typing human platelet or red blood cell surfacemembrane glycoproteins, comprising the steps of (A) synthesizing cDNAfrom human platelet or red blood cell mRNA of a first individual, (B)amplifying the cDNA to produce amplified cDNA and (C) analyzing theamplified cDNA to determine a platelet or red blood cell phenotype. Inone preferred embodiment, step (C) comprises

(i) digesting the amplified cDNA with a restriction endonuclease toproduce cDNA fragments, the endonuclease recognizing a cleavage sitethat distinguishes a nucleotide sequence of an allele of a platelet orred blood cell alloantigen from other alleles and (ii) analyzing thecDNA fragments to determine a platelet or red blood cell phenotype. Inanother preferred embodiment, the cDNA comprises a sequence that isencoded by at least nucleotide 196 of GPIIIa mRNA and the restrictionendonuclease recognizes a restriction site, which comprises or isadjacent to a nucleotide sequence that includes nucleotide 196 ofGPIIIa.

A method has also been provided, in accordance with still another aspectof the present invention, for typing platelet Pl^(A) surface membraneglycoproteins comprising the steps of (A) obtaining genomic DNA from afirst individual and (B) analyzing the genomic DNA to determine aplatelet Pl^(A) alloantigen phenotype. In a preferred embodiment, step(B) comprises (i) digesting the genomic DNA with a restrictionendonuclease to produce DNA fragments, (ii) hybridizing the DNAfragments with a labeled allele-specific oligonucleotide probe thatdistinguishes a nucleotide sequence of an allele of a platelet Pl^(A)alloantigen from other alleles and (iii) analyzing the DNA fragmentsthat have hybridized to the probe to determine a platelet PlA phenotype.

According to another aspect of the present invention, a method isprovided for typing a platelet Pl^(A) alloantigen comprising the stepsof (A) obtaining genomic DNA from a first individual, (B) amplifying thegenomic DNA to produce amplified genomic DNA and (C) analyzing theamplified genomic DNA to determine a platelet Pl^(A) alloantigenphenotype. Step (C), in one preferred embodiment, comprises (i)hybridizing the amplified genomic DNA with a pair of oligonucleotideprobes, wherein a first probe of the pair of probes is labeled with afirst probe and a second probe of the pair of probes is labeled with asecond probe, such that the first probe is distinctly labeled from thesecond probe, such that the pair of probes hybridize adjacently to eachother at a nucleotide, wherein the nucleotide distinguishes an allele,of a platelet Pl^(A) alloantigen, from other alleles, (ii) ligating theprobes in a ligation reaction to produce ligation products, wherein (a)the first probe becomes ligated to the second probe when adjacentnucleotides of the first and the second probe both hybridize to theamplified genomic DNA or (b) the first probe and the second probe arenot ligated and (iii) analyzing the products of the ligation reaction todetermine the alloantigen phenotype.

In accordance with another aspect of the present invention, there hasbeen provided polypeptide sequence encoded by a nucleotide sequencecomprising a polynucleotide encoding at least amino acid 33 of GPIIIa,that distinguishes platelet membrane glycoprotein Pl^(A1) from Pl^(A2).

There has also been provided, in accordance with still another aspect ofthis invention, an antibody that binds the Pl^(A1) allele of GPIIIa orthe Pl^(A2) allele of GPIIIa, (i) the antibody recognizing a polypeptidemolecule encoded by a nucleotide sequence comprising an oligonucleotideencoding at least amino acid 33 of GPIIIa that distinguishes plateletmembrane glycoprotein Pl^(A1) from Pl^(A2) and (ii) the antibody bindingeither the Pl^(A1) form or the Pl^(A2) form.

According to still another aspect of the present invention, a method isprovided for treating post-transfusion purpura or neonatal alloimmunethrombocytopenia (NATP), comprising the step of administering to a firstindividual a formulation comprised of a peptide in a pharmacologicallyeffective concentration and a physiologically-compatible carriertherefor, the first individual suffering from the post-transfusionpurpura or the mother of a fetus at risk for developing NATP, and havinganti-Pl^(A1) or anti-Pl^(A2) antibodies, wherein the peptide binds anantibody selected anti-Pl^(A2) antibody.

There has also been provided, according to a further aspect of thepresent invention, a kit for typing platelet Pl^(A) alloantigens,comprising (a) a receptacle containing a solution of an endonucleaserecognizing a cleavage site that distinguishes a nucleotide sequence ofan allele of a platelet PlA alloantigen from other alleles, (b) areceptacle containing a solution of a labeled oligonucleotide probe thatdistinguishes an allele of a platelet Pl^(A) alloantigen, the allelecomprising nucleotide 196 of GPIIIa, from other alleles, (c) areceptacle containing a solution of an antibody that binds a Pl^(A1)antigen or a Pl^(A2) antigen, the antibody (i) recognizing a polypeptidemolecule encoded by a nucleotide sequence encoding at least amino acid33 of GPIIIa and (ii) binding either the Pl^(A1) antigen or the Pl^(A2)antigen.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is diagrammatic representation of the NH₂ -terminal region of theGPIIIa mRNA molecule. The locations of two oligonucleotide primers usedfor PcR amplification are also shown, as is the polymorphic sequence atbase 196 of the amplified cDNA.

FIG. 2 is a autoradiograph of an electrophoretic gel used in thesequence analysis of amplified GPIIIa cDNA, derived from a Pl^(A2)homozygous individual. A segment of the autoradiograph, encompassingbases 188-203, is shown. A single base substitution of a cytosine (C)(Pl^(A2) allele) for a thymidine (T) (Pl^(A1) allele) at base 196 isindicated with an arrow.

FIG. 3 is a photograph of an electrophoretic gel generated in the courseof analyzing Pl^(A) phenotype by restriction endonuclease digestion, inaccordance with the present invention. Bases 56-408 were enzymaticallyamplified from platelet RNA from eighteen individuals of known Pl^(A)phenotype. The results from three representative individuals of eachpossible phenotype are shown on this gel. Heterozygotes for the alleleshow three bands, corresponding to undigested (Pl^(A1) -derived) andcleaved (Pl^(A2) -derived) GPIIIa cDNA.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Although the generation of cDNA from platelet or red blood cell mRNA waspreviously thought to be unfeasible, a new approach has been discoveredfor examining platelet- and red blood cell-specific mRNA sequences fromsingle individuals. It is been found that mRNA can be obtained fromplatelets and red blood cells in quantities sufficient for isolation,cDNA generation, and amplification. By generating and amplifying cDNAproduced from mRNA of a number of individuals of known red blood cell orplatelet allotypes, the nucleotide sequence variations that exist in thegenes that express alloantigen determinants can be ascertained.Furthermore, by isolating and amplifying mRNA from a number ofindividuals of known allotype, it is possible, pursuant to the presentinvention, to identify phenotype-specific nucleotide sequence variationsin corresponding genes.

To obtain amplified cDNA from red blood cell or platelet mRNA, mRNAderived, using conventional methods, see, e.g., MANIATIS ET AL,MOLECULAR CLONING: A LABORATORY MANUAL 187-210 (Cold Spring HarbourLaboratory, 1982), from platelets or red blood cells can be converted tocDNA and then enzymatically amplified to produce microgram quantities ofplatelet- or red blood cell-specific cDNA. This amplification ispreferably accomplished via the "polymerase chain reaction" (PcR) methoddisclosed by U.S. Pat. Nos. 4,683,195 and 4,800,159, the respectivecontents of which are hereby incorporated by reference.

More specifically, in the process of generating and amplifying cDNAencoded by the isolated platelet or red blood cell mRNA, oligonucleotideprimer pairs can be constructed that allow enzymatic amplification of asegment of an mRNA molecule that encodes an amino-acid sequence definingthe polymorphism. The corresponding, isolated cDNAs can then be analyzedto determine the molecular basis of observed phenotypic differences. Theability to compare directly the nucleotide and corresponding amino-acidsequences of genes encoding alleles of alloantigens is made possible by(1) the discovery that cDNA can be generated and amplified successfullyfrom platelet or red blood cell mRNAs and (2) the determination of anucleotide sequence of an alloantigen which is thought to bepolymorphic. As such, this application represents a novel application ofthe PcR technique heretofore not deemed feasible.

The molecular description of polymorphisms associated with platelet orred blood cell alloantigens can be provided by analyzing amplified cDNA,generated from red blood cell or platelet mRNA, according to one of thefollowing methods: differential restriction endonuclease digestion(DRED), allele-specific oligonucleotide probing (ASOP), andligase-mediated gene detection (LMGD). Additional methods of analysiswould also be useful in this context, such as fluorescence resonanceenergy transfer (FRET) as disclosed by Wolf et al, Proc. Nat. Acad. Sci.USA 85: 8790-94 (1988), the contents of which are hereby incorporated byreference.

DRED analysis is accomplished in the following manner. If conditionsoccur including (1) a particular amplified cDNA segment contains asequence variation that distinguishes an allele of a polymorphism and(2) this sequence variation is recognized by a restriction endonuclease,then the cleavage by the enzyme of a particular polynucleotide segmentcan be used to determine the alloantigen phenotype. In accomplishingthis determination, amplified cDNA derived from platelet or red bloodcell mRNA is digested and the resulting fragments are analyzed by size.The presence or absence of nucleotide fragments, corresponding to theendonuclease-cleaved fragments, determines which phenotype is present.

For example, cDNA generated from GPIIIA mRNA and acytosine(C)←→thymidine(T) polymorphism at base 196 is revealed byexamination of the nucleotide sequence contained in cDNA generated frommRNA derived from a Pl^(A2) homozygous individual. This singlenucleotide substitution results in the creation of a unique restrictionenzyme cleavage site for the restriction endonuclease NciI. In thisexample, utilizing the ability of the restriction endonuclease NciI, todiscriminate between these two polymorphic sequences, the phenotypes ofindividuals can be determined by the above described manner. Then, afterfurther sequence analysis of the resulting restriction fragments, it canbe demonstrated that the Pl^(A2) form of GPIIIa mRNA contains the codonCCG (encoding the amino acid proline at position 33), in place of theCUG (which encodes leucine at position 33), present in the Pl^(A1)allele.

In ASOP analysis according to conventional methods, oligonucleotideprobes are synthesized that will hybridize, under appropriate annealingconditions, exclusively to a particular amplified cDNA segment thatcontains a nucleotide sequence that distinguishes one allele from otheralleles of a red blood cell or platelet membrane glycoprotein. Thisspecific probe is discernably labeled so that when it hybridizes to theallele distinguishing cDNA segment, it can be detected, and the specificallele is thus identified.

For example, oligonucleotide probes can be synthesized, in accordancewith the present invention, that will hybridize to a cDNA segmentencoded by GPIIIa mRNA that contains either cytosine or thymidine atnucleotide 196, but not both. These allele-specific probes can then belabeled appropriately and added to the generated cDNA segments underannealing conditions, such that one of the allele-specific probeshybridizes and can be detected. The specific Pl^(A1) or Pl^(A2) allelesequence is thus identified.

In the course of the third method of analysis, LMGD, as disclosed byLandegren et al., Science 241: 1077-80 (1988), the contents of which arehereby incorporated by reference, a pair of oligonucleotide probes aresynthesized that will hybridize adjacently to each other, i.e., to acDNA segment under appropriate annealing conditions, at the specificnucleotide that distinguishes one allele from other alleles of a redblood cell or platelet membrane glycoprotein. Each of the pair ofspecific probes is labeled in a different manner, and when it hybridizesto the allele-distinguishing cDNA segment, both probes can be ligatedtogether by the addition of a ligase.

When the ligated probes are isolated from the cDNA segments, both typesof labeling can be observed together, confirming the presence of theallele-specific nucleotide sequence. Where the above- described pair ofdifferently labeled probes bind to a nucleotide sequence containing adistinguishing nucleotide of a different allele, the probe pair is notligatable and, after the probes are isolated from the cDNA segments,both types of labeling are observed separately.

An exemplary LMGD analysis, according to the present invention, entailsthe use of a pair of oligonucleotide probes that bind to a cDNA segmentadjacently to each other at a nucleotide corresponding to nucleotide 196of GPIIIa, wherein one probe is radioactively 32P-labeled and the otherprobe is biotin-labeled. The biotin labeled probe hybridizes nucleotides176-196 of GPIIIa, wherein nucleotide 196 contains a cytosine, whichdistinguishes the Pl^(A2) allele. The ³² P labeled probe hybridizesnucleotides 197-217 of GPIIIa and, therefore will hybridize adjacentlyto the biotin labeled probe. These probes are then added under annealingconditions such that they hybridize adjacently to each other spanningnucleotides 176-217 of GPIIIa. When the Pl^(A2) allele sequence ispresent in the amplified cDNA, then the addition of a ligase will resultin the biotin labeled probe being covalently bound to the ³² P-labeledprobe. The ligation is possible, because the ends of the probes that areadjacent to each other (hybridized to nucleotides 196 and 197) are bothhybridized to the cDNA. In the case where these two probes hybridize tothe Pl^(A1) allelic form of the cDNA sequence, the biotin-labeled probeend at nucleotide 196 is not hybridized appropriately, preventing theligation step from occurring. When this pair of probes binds completelyto the Pl^(A2) allele sequence, therefore, the probes are ligated andwhen the probes are separated from the Pl^(A2) sequence and exposed soas to be detected, both the biotin/strepavidin and the ³² P labeling arepresent together. When the Pl^(A1) allele sequence is hybridized, on theother hand, the probes cannot be ligated, and the biotin/strepavidin-and³² P-labeling are observed separately. In this manner, the Pl^(A1) andPl^(A2) alleles sequences and corresponding phenotype can bedistinguished.

Alternatively, DRED, ASOP and LMGD or other suitable methods ofanalysis, such as FRET, can be used with genomic or amplified-genomicDNA to distinguish platelet membrane glycoprotein Pl^(A1) from Pl^(A2),starting with any nucleated cell sample, obtained from an individual,from which DNA can be isolated in sufficient quantities for analysis.Amplified genomic DNA would be amplified from isolated genomic DNA inthe same manner as described above for cDNA. Once a tissue sample, suchas cells scraped from the inside of an individual's cheek, has beenobtained, genomic DNA isolated by conventional procedures can beanalyzed directly per se or amplified prior to analysis.

The foregoing description of the three types of analysis would apply tothe use of genomic DNA or amplified-genomic DNA, with the term "cDNA"being replaced with "genomic or amplified genomic DNA." One differencein the analysis of genomic DNA or amplified genomic DNA is that theGPIIIa sequence used for designing a suitable oligonucleotide probewould have to include any intronic sequences, which would not be presentin the cDNA of GPIIIa, that were near or adjacent to the nucleotide thatdetermines the Pl^(A) phenotype.

The presence of intronic sequences near the phenotype-determiningnucleotide can be determined by sequence analysis of genomic DNA,accomplished via Maxam-Gilbert or another conventional technique.Sequence-analysis data which indicate that an intron is near thephenotype-determining nucleotide can be used to design oligonucleotidesthat span the intron/exon boundary, so that a Pl^(A) phenotype can beascertained.

For example, sequence analysis conducted by Dr. Gilbert C. White II,University of North Carolina, (Chapel Hill, NC), has shown that an800-nucleotide intron is located between bases 185 and 186 of the GPIIIacDNA (personal communication of unpublished results). A nucleotidesequence is set out below which includes the intron/exon boundary andwhich is relevant to the design of a suitable, Pl^(A)phenotype-determining oligonucleotide probe:

    5,'-tttgggctcctgtcttacag/GCCCTGCCT-3,'

In this sequence, the lower case letters represent the intronic sequenceand the upper case letters represent the cDNA sequence. The linerepresents the intron/exon boundary.

The ability to perform DNA-typing analysis for determination of Pl^(A)phenotypes, pursuant to the present invention, has a number of usefulclinical applications, including but not limited to those involvingdetermination of an alloantigen phenotype of an individual, and thediagnosis and treatment of a pathological immune response (or potentialresponse) involving foreign alloantigens or antibodies. In accordancewith the present invention, alloantigen phenotyping is effected bygenerating amplified cDNA from red blood cell or platelet mRNA, whichpermits diagnosis of individuals for the purpose of treating orpreventing pathological immune responses. Once the nucleotide-sequencevariations specific for each allelic form of the alloantigens of a givenclass are determined, other oonventional methods can be employed,through the use of genomic DNA or platelet RNA, to perform the same typeof diagnosis on other individuals. These methods would include, but arenot limited to, allele-specific nucleotide probing and ligase-mediatedgene detection, as previously described.

Diagnostic kits can also be used, in accordance with the presentinvention, for the determination and diagnosis of alloantigen phenotypes

via the procedures described herein. Such a kit can include, inter alia,antibodies to alloantigens expressed by the above-described Pl^(Al) -and Pl^(A2) -encoding sequences, which antibodies would react with theblood sample of an individual so as to indicate whether that individualhas a Pl^(A1) or Pl^(A2) phenotype. Alternatively, all the reagentsrequired for the detection of nucleotide(s) that distinguish the Pl^(A)alloantigens, by means described herein, can be provided in a single kitthat uses isolated genomic DNA or platelet mRNA from an individual. Sucha kit, containing a restriction endonuclease and/or a labeled probe thatdistinguish nucleotide 196 of GPIIIa, can be utilized for PlAalloantigen phenotyping.

A further beneficial use of the nucleotide sequences that distinguishthe Pl^(A1) allele from the Pl^(A2) allele is to obtain or synthesizethe respective expression product, in the form of a peptide, encoded bythese nucleotide sequences. These peptides can be used to generateantibodies for diagnostic and therapeutic uses, for example, with regardto pathological conditions such as PTP or NATP.

In this context, the term "antibodies" is used to denote, inter alia,monoclonal or polyclonal antibodies, specific for peptides generatedfrom nucleotide sequences distinguishing either Pl^(A1) or Pl^(A2)alleles, that can be generated by injecting a peptide into mice or othersuitable laboratory animal. In the case of monoclonal antibodyproduction, one proceeds by isolating lymphocytes and fusing them withmyeloma cells, producing hybridomas. The cloned hybridomas are thenscreened for production of antibodies specific for sequences that arediagnostic of the Pl^(A1) or Pl^(A2) form of the GPIIIa membraneglycoprotein. In addition, the term "antibodies" encompasses fragments,like Fab and F(ab')₂, of anti-Pl^(A1) or anti-Pl^(A2) antibodies, andconjugates of such fragments, and so-called "antigen binding proteins"(single-chain antibodies) based on anti-Pl^(A1) or anti-Pl^(A2)antibodies, in accordance, for example, with U.S. Pat. No. 4,704,692,the contents of which are hereby incorporated by reference. Humanalloantisera currently used for serological typing are specificallyexcluded from this definition.

Diagnostic applications of these antibodies are exemplified, accordingto the present invention, by the use of a kit containing anti-Pl^(A1) oranti-Pl^(A2) antibcdies which undergo a reaction with a sample of anindividual's blood to determine a Pl^(A1) or Pl^(A2) platelet phenotype.Such a reaction involves the binding of anti-Pl^(A1) antibodies toPl^(A1) antigens or the binding of of antibody-antigen complexes in ablood sample would indicate a positive result. A kit of this sort couldbe used to diagnose, or to help prevent, the occurrence of pathologicalconditions like PTP or NATP.

Therapeutic applications for the peptides encoded by these Pl^(A1)/Pl^(A2) -distinguishing nucleotide sequences, and the correspondinganti-Pl^(A1) or anti-Pl^(A2) antibodies, include their use in thegeneration, via conventional methods, of antiidiotypic antibodies (thatis, "anti-" anti-Pl^(A1) or anti-Pl^(A2) antibodies) such as fromhybridomas, as described above. See, for example, U.S. Pat. No.4,699,880, the contents of which are hereby incorporated by reference.Such antiidiotypic antibodies would bind endogenous or foreignanti-Pl^(A) antibodies in the blood of an individual, thereby to treator prevent pathological conditions associated with an immune response toa "foreign" Pl^(A) alloantigen.

The present invention is further described below by reference to thefollowing, illustrative examples. Used in the examples were plasmasamples from ten homozygous Pl^(A1) individuals, five homozygous Pl^(A2)individuals, and three individuals who were heterozygous for the Pl^(A)allotype. The respective phenotypes of all the test subjects had beenidentified using well-characterized anti-Pl^(A1) and anti-Pl^(A2) humanalloantisera.

EXAMPLE 1 Amplification of Platelet mRNA

In order to examine the amino terminal region of the Pl^(A2) allele, thepolymerase chain reaction was used to amplify a 366 base pair (bp)region near the 5, end of platelet GPIIIa mRNA. Human platelets wereisolated from whole blood as previously described (Newman et al., Blood69:668-676 (1987)). Briefly, 50 ml of venous blood collected fromhealthy volunteer donors was anticoagulated with 7 ml of acid citratedexrose (National Institutes of Health, Formula A), and PGE₁ was addedto a final concentration of 50 nmol/liter. Platelet-rich plasma (PRP)was prepared by differential centrifugation, and the platelets presentin the upper half of the PRP were harvested and washed an additional twotimes in wash buffer (100 mmol/liter NaCl, 8.5 mmol/liter Tris, 8.5mmol/liter glucose, and 1 mmol/liter Na₂ EDTA, pH 7.4). By carefullyavoiding the lower half o the PRP, consistent achievement of aplatelet/leukocyte ratio of at least 3000:1 was attained. The finalpaltelet pellet was solubilized in 3.3 ml of buffer composed of 4mol/liter guanidinium isothiocyanate, 5 mmol/liter sodium citrate, 0.1mol/liter 2-mercaptoethanol, 0.5% SARKOSYL, 0.1% ANTIFOAM A, and 40 U/mlplacental RNAse inhibitor, layered onto a 1.2 ml cushion of 5.7mol/liter CsCl, and centrifuged at 20° C. in a Ti50.1 rotor at 35,000rpm for 16 hours (Beckman Instruments, Inc., Fullerton, Calif.). Thenearly invisible RNA pellet was resuspended in buffer made up of 10mmol/liter Tris, 5 mmol/liter EDTA, and 1% SDS, Ph 7.4), extracted withchloroform/butanol, and ethanol precipitated essentially as described byManiatis et al., loc. cit. The resulting purified total platelet RNAfraction recovered using this technique was <1 μg per individual, andwas used in all subsequent steps without further fractionation. Ouramplification strategy employed 2 sets of oligonucleotide primers, onenested internally to the first. Nested primers are not always necessary.

Two pairs of primers were constructed; an outer pair (Primers 1 and 3),and an internally nested pair (Primers 2 and 4) and used to amplify aregion of the GPIIIa mRNA molecule that encodes the amino terminus ofmature glycoprotein IIIa. Primer (5'-CGCGGGAGGCGGACGAGATGCG-3')corresponds to the RNA strand from bases 4-25 of the publishednucleotide sequence (Fitzgerald et al., J. Biol. Chem. 262: 3936-3939,987). Primer 2 (5'-GACTCGAGACTGTGCTGGCGCTG-3') corresponds to bases56-71 of the RNA strand, with an additional 7 bp encompassing an Xho Irestriction enzyme recognition site incorporated onto the 5'-end tofacilitate subsequent subcloning into plasmid vectors. The twoanti-sense oligonucleotides were Primer 3(5'-CGCACT-TGGATGGAGAAATTC-3'), which corresponds to nucleotides 412-392, and Primer 4 (5'-CCGGATCCTTGGATGGAGAAATTC-3'), which correspondsto bases 408-392 plus an additional 7 base pairs that contain a Bam HIsite. Primer 3 was used to prime first strand cDNA synthesis, usingplatelet mRNA as a template, in a total volume of 50 μl as previouslydescribed, id. Newman et al (1988). All ensuing PcR reactions Wereperformed in a programmable DNA Thermal Cycler (Perkin-Elmer CetusCorp., Norwalk, Conn.).

The first 10 rounds of PcR were performed using Primers 1 and 3 in atotal volume of 100 μl using a regimen consisting of denaturingpolynucleotide strands at 94° C. for 90 sec., annealing primers at 37°C. for 2 min, and primer extending with Taq polymerase (Perkin-ElmerCetus Corp.) at 72° C. for 3.5 minutes. Following the fifth thermalcycle, the primer annealing temperature was increased to 42° C. Afterthe tenth cycle, the first primer pair was removed by centrifuge-drivendialysis of the PcR reaction mixture into a nearly identical bufferusing Centricon 30 microconcentrators (Amicon Corp, Danvers, Mass.). Thefirst 10 thermal cycles of the PcR amplified bases 4-412 approximatelyone-thousand fold, and provided a sufficient quantity of GPIIIa-specificcDNA to permit more stringent conditions to be used in subsequentrounds.

The second reaction mix was identical to the first, except thatinternally nested Primers 2 and 4 were used in place of Primers and 3.Following oligonucleotide exchange, the reaction volume was againbrought to 100 μl including 2.5 units of fresh Taq polymerase, and PcRcontinued for an additional 21 thermal cycles. Primer annealing wasperformed at 42° C. for rounds 11-15; 47° C. for rounds 16-20, and 55°C. for rounds 21-31. It was found that these conditions maximizedspecificity and yield for amplification of this particular cDNA. Thepresence of the additional bases used to form the restriction enzymesites at the 5' ends of Primers 2 and 4 had no detrimental effect on thequantity of specific DNA produced during the PcR. The remaining thermalcycles amplified bases 56-408 using an internally nested primer pair,which are graphically depicted in the top portion of FIG. 1. This regionencodes the first 103 amino acids of the mature GPIIIa protein, as wellas a majority of the signal peptide. Using this protocol, we producedmicrogram amounts of the expected 366 bp cDNA from a number ofindividuals of known Pl^(A) phenotype.

EXAMPLE 2 Sequence Analysis of Amplified cDNAs

Selected amplified cDNA's were subcloned into the plasmid vectorpGEM-7Zf (Promega Biotech, Madison, Wis.), and subjected to nucleotidesequence analysis. Dideoxy-sequencing was performed using the modifiedT7 phage DNA polymerase, Sequenasetm (United States Biochemicals,Cleveland, Ohio), according to the manufacturer's directions. Thecomplete nucleotide sequence of bases 79-408 from one Pl^(A2) homozygousindividual was determined on both strands, and found to be identical tothe 3 previously reported sequences for GPIIIa, except at base 196(sequence numbering according to reference #6), which had adeoxycytosine (C) in place of a deoxythymidine (T). Sequence analysis of2 different sub-clones of the 366 bp Pl^(A2) -derived cDNA productshowed the same base substitution, making it unlikely that thisnucleotide difference was an artifact generated in vitro during eitherthe reverse transcriptase or Taq polymerase reactions. This single basechange (shown in FIG. 2) results in substitution of a Proline forLeucine at amino acid residue 33 of the mature GPIIIa molecule, and islikely to impart significant secondary structural differences in thepolypeptide chain.

EXAMPLE 3 Restriotion Enzyme Analysis of GPIIIa Allotypes.

Analysis of the GPIIIa sequence from bases 56-408 revealed thatsubstitution of a C for a T at base 196 would create a recognition sitefor the restriction enzyme NciI, which cleaves at 5'-CCGGG-3, but not5'-CTGGG-3' sequences. To determine whether the thymidine (T)→cytosine(C) substitution found in the Pl^(A2) individual studied above wasrelated to Pl^(A) allotype, or merely represented an unrelatedpolymorphism of GPIIIa, platelet RNA was prepared from a total of 18individuals of known Pl^(A) phenotype, and then amplified using PcR toyield the same 366 bp product. As illustrated in FIG. 1, NciI should notbe able to cleave the 366 bp cDNA from a Pl^(A1) homozygous individual,whereas 2 fragments of 147 and 219 bp would be generated in a Pl^(A2)homozygote.

Most PcR reaction products were directly excnanged into sterile waterusing Centricon 30 microconcentrators, and then digested with NciI(purchased from either New England Biolabs, Beverly, Mass., or BethesdaResearch Laboratories, Gaithersburg, Md.). Restriction digests wereanalyzed on 1.5% agarose gels. Computer analyses of protein andpolynucleotide sequences were performed using the program PC/GENE(Intelligenetics Inc . Mountain View, Calif.) operating on an IBMPC/AT-compatible personal computer. FIG. 3 shows that the 366bp cDNAamplified from Pl^(A1) homozygous individuals was not cleaved by NciI,whereas the predicted 219- and 147-bp restriction fragments wereobtained from Pl^(A2/A2) -derived cDNA. Serologically-determinedheterozygotes for the Pl^(A) phenotype, containing both the Pl^(A1) andPl^(A2) cDNA sequences, yielded both uncut, 366-bp cDNA and the 219- and147-bp restriction fragments.

The DNA-typing procedure of the present invention, as described above,predicted the phenotype of ten Pl^(A1/A1), three Pl^(A1/A2), and fivePl^(A1/A2) individuals (ρ=3.8×10⁻⁶).

What is claimed is:
 1. A method of typing human platelet glycoproteins,comprising the steps of(a) synthesizing cDNA from human platelet; (b)amplifying said cDNA to produce amplified cDNA; and then (c) analyzingsaid amplified cDNA to determine a paltelet phenotype.
 2. A method asclaimed in claim 1, further comprising the step of synthesizing cDNAfrom human platelet of a second individual, and then repeating steps (b)and (c) on the CDNA of said second individual; and thereafterdiscriminating between said first and second individuals based on saidalloantigen phenotype.
 3. A method as claimed in claim 1, wherein step(c) comprises the steps of (i) digesting said amplified cDNA wtih arestriction endonuclease recognizing a cleavage site that distinguishesa nucleotide sequence of an allele, of a platelet alloantigen, fromother alleles; and analyzing said cDNA fragments to determine a plateletphenotype.
 4. A method as claimed in cailm 3 wherein (i) said cDNAcomprises a sequence that encodes at least nucleotide 196 of CPIIIa mRNAand (ii) said restriction endonuclease recognizes a restriction site,which comprises or is adjacent to a nucleotide sequence that includesnucleotide 196 of CPIIIa mRNA.
 5. A method as claimed in claim 1,wherein step (c) comprises the steps of (i) hydridizing said cDNAfragments with a labeled allele-specific oligonucleotide probe thatdistinguishes a nucleotide sequence of a Pl^(A) allele from anotherPl^(A) allele; and then (ii) analyzing said probe that has hybridized tosaid cDNA fragments to determine said Pl^(A) alloantigen phenotype.
 6. Amethod as claimed in claim 5, wherein said Pl^(A) allele comprises asequence that is encoded by at least nucleotide 196 of CPIIIa mRNA andsaid labeled oligonucleotide probe hybridizes to a nucleotide sequencewhich comprises or is adjacent to nucleotide 196 of GPIIIa mRNA.
 7. Amethod as claimed in claim 1, wherein step (c) comprises the steps of(i)hybridizing said amplified cDNA with a pair of oligonucleotide probes toproduce a construct, wherein a first probe of said pair is labeled witha first label and a second probe of said pair is labeled with a secondlabel, such that the first label is distinguishable from the secondlable, and the probes hydbridize adjacent to each other at a nucleotidethat distinguishes a first Pl^(A) allele from another Pl^(A) allele;thereafter (ii) reacting said construct with a ligase in a reactionmedium; and then (iii) analyzing said reaction medium to detect thepresence of a ligation product comprising said first probe and saidsecond probe.
 8. A method as claimed in claim 7, wherein said Pl^(A)allele comprises a sequence that is encoded by at least nucleotide 196of GPIIIa mRNA and said labeled oligonucleotide probes hybridize anucleotide sequence which comprsies or is adjacent to nucleotide 196 ofGPIIIa mRNA.
 9. A method of typing platelet Pl^(A) membraneglycoproteins comprising the steps of (a) obtaining genomic DNA from afirst individual and (b) analyzing said genomic DNA to determine aplatelet Pl^(A) alloantigen phenotype.
 10. A method as claimed in claim9, further comprising the step of obtaining genomic DNA from a secondindividual, and then repeating step (b) on the genomic DNA of saidsecond individual; and thereafter discriminating between said first andsecond individuals based on said Pl^(A) alloantigen phenotype.
 11. Amethod as claimed in claim 9, whrein step (b) comprises the steps of (i)digesting said genomic DNA with a restriction endonuclease to produceDNA fragments, said endonuclease recognizing a cleavage site thatdistinguishes a nucleotide sequence of a first Pl^(A) allele from thatof another Pl^(A) allele; and then (ii) analyzing said DNA fragmentswith a labeled probe to determine said Pl^(A) alloantigen phenotype. 12.A method as claimed in claim 11, wherein said Pl^(A) allele comprises asequence that is encoded by at least nucleotide 196 of GPIIIa mRNA andsaid restriction endonuclease recognizes a restriction site whichcomprises or is adjacent to a nucleotide sequence that includesnucleotide 196 of GPIIIa mRNA.
 13. A method as claimed in claim 9,wherein step (b) comprises the steps of (i) digesting said genomic DNAwith a restriction endonuclease to produce DNA fragments; thereafter(ii) hybridizing said DNA fragments with a labeled allele-specificoligonucleotide probe that distinguishes a nucleotide sequence of anallele of a platelet Pl^(A) alloantigen from other alleles; and then(iii) analyzing said probe that has hybridized to said DNA fragments todetermine said Pl^(A) alloantigen phenotype.
 14. A method as claimed inclaim 13, wherein said Pl^(A) allele comprises a sequence that isencoded by at least nucleotide 196 of GPIIIa mRNA and said labeledoligonucleotide probe hybridizes to a nucleotide sequence whichcomprises or is adjacent to nucleotide 196 of GPIIIa mRNA.
 15. A methodas claimed in claim 9, wherein step (b) comprises the steps of (i)hybridizing said genomic DNA with a pair of oligonucleotide probes toproduce a construct, wherein a first probe of said pair is labeled witha first label and a second probe of said pair is labeled with a secondlabe, such that the first label is distinguishable from the secondlabel, and the pair of probes hybridize adjacently to each other at anucleotide of said genomic DNA that distinguishes a Pl^(A) allele fromanother Pl^(A) allele; thereafter (ii) reacting said construct with aligase in a reaction medium; and then (iii) analyzing said reactionmedium to detect the presence of a ligation product comprising saidfirst probe and said second probe.
 16. A method as claimed in claim 15,wherein said Pl^(A) allele comprises a sequence that is encoded by atleast nucleotide 196 of GPIIIa mRNA and said labeled oligonucleotideprobes hybridize to a nucleotide sequence which comprises or is adjacentto nucleotide 196 of GPIIIa mRNA.
 17. A method of typing platelets withrespect to GPIIIA comprising the steps of (a) obtaining genomic DNA froman first individual, (b) amplifying said genomic DNA to produceamplified genomic DNA and (c) analyzing said amplified genomic DNA todetermine a platelet Pl^(A) alloantigen phenotype.
 18. A method asclaimed in claim 17, further comprising the step of obtaining genomicDNA from a second individual, and then repeating steps (b) and (c) onthe genomic DNA of said second individual; and thereafter discriminatingbetween said first and second individuals based on said alloantigenphenotype.
 19. A method as claimed in claim 17, wherein step (c)comprises the steps of (i) digesting said amplified genomic DNA with arestriction endonuclease to produce DNA fragments, said endonucleaserecognizing a cleavage site that distinguishes a nucleotide sequence ofa first Pl^(A) allele fro mthat of another Pl^(A) allele; and thereafter(ii) analyzing said DNA fragments to determine said alloantigenphenotype.
 20. A method as claimed in claim 17, wherein step (c)comprises the steps of (i) hybridizing said amplified genomic DNA with alabeled allele-specific oligonucleotide probe that distinguishes anucleotide sequence of a Pl^(A) allele from that of another Pl^(A)allele; and then (ii) analyzing said probe that has hybridized to saidamplified genomic DNA to determine said alloantigen phenotype.
 21. Amethod as claimed in claim 20, wherein said Pl^(A) allele comprises asequence that is encoded by at least nucleotide 196 of GPIIIa mRNA andsaid labeled oligonucleotide probe hybridizes to a nucleotide sequencewhich comprises or is adjacent to nucleotide 196 of GPIIIa mRNA.
 22. Amethod as claimed in claim 17, wherein step (c) comprises the steps of(i) hybridizing said amplified genomic DNA with a pair ofoligonucleotide probes to form a construct, wherein a first probe ofsaid pair is labeled with a first label and a second probe of said pairis labeled with a second label, such that the first label isdistinguishable from the second label, and the probes hybridizeadjacently to each other at a nucleotide that distinguishes a Pl^(A1)allele from another a Pl^(A2) allele; thereafter (ii) reacting saidconstruct with a ligase in a reaction medium; and then (iii) analyzingsaid reaction medium to detect the presence of a ligation productcomprising said first probe and said second probe.
 23. A method asclaimed in claim 19, wherein said Pl^(A) allele comprises a sequencethat is encoded by at least nucleotide 196 of GPIIIA mRNA and saidlabeled oligonucleotide probe hybridizes to a nucleotide sequence, whichcomprises or is adjacent to nucleotide 196 of GPIIIA mRNA.
 24. A kit fortyping platelet Pl^(A) alloantigens, comprising(a) means for amplifyingDNA that comprises at least a portion of a GPIIIA gene or GPIIIA cDNA,wherein said portion includes a nucleotide corresponding to nucleotide196 of said GPIIIA cDNA, and at least one component selected from thegroup consisting of (b) a receptacle containing a solution of a labeledoligonucleotide probe that distinguishes a Pl^(A) allele from anotherPl^(A) allele, and (c) a receptacle containing a solution of anendonuclease recognizing a cleavage site that distinguishes a nucleotidesequence of a Pl^(A) allele from another Pl^(A) alleles.
 25. A kit fortyping platelet Pl^(A) alloantigens, comprising a receptacle containinga solution of an antibody that discriminately binds alloantigensexpressed by a Pl^(A1) allele or a Pl^(A2) allele of GPIIIA, saidantibody (i) recognizing a peptide molecule encoded by a nucleotidesequence encoding at least amino acid 33 of GPIIIA and (ii) bindingeither said Pl^(A1) allele or said Pl^(A2) allele of GPIIIA.
 26. Amethod as claimed in claim 22, wherein said Pl^(A) allele comprises asequence that is encoded by at least nucleotide 196 of GPIIIa mRNA andsaid labeled oligonucleotide probe hybridizes to a nucleotide sequencewhich comprises or is adjacent to nucleotide 196 of GPIIIa mRNA.